Biochip for the detection of phosphorylation and the detection method using the same

ABSTRACT

The present invention relates to a biochip for the detection of phosphorylation and a detection method of phosphorylation using the same, more precisely a biochip prepared by integrating a recombinant fusion protein produced from the reaction of a kinase matrix selected from the group consisting of PKC (Protein Kinase C), cc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase) and the elevated protein Selenomonas ruminantium membrane protein on a substrate coated with an active group, a detection kit of phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope and a detection method of phosphorylation using the same. The biochip for the detection of phosphorylation of the present invention using a radio-isotope facilitates the detection of phosphorylation with a minimum amount of a sample by simple processes, compared with the conventional method using an antibody. Since this method can analyze a large amount of samples in a shorter period of time, it can be effectively used for the analysis of kinase activity.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a biochip for the detection of phosphorylation and a detection method of phosphorylation using the same, more precisely a biochip prepared by integrating a recombinant fusion protein produced from the reaction of a kinase matrix selected from the group consisting of PKC (Protein Kinase C), cc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase) and the elevated protein Selenomonas ruminantium membrane protein on a substrate coated with an active group, a detection kit of phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope and a detection method of phosphorylation using the same.

2. Description of the Related Art

Recent biotechnology industry is characterized by the fusion of biology with related technologies in the fields of electronics, computer science and mechanical engineering. Biological studies require a new system to approach an individual biological subject as a whole and also require a novel approaching method facilitating experiments in a large scale with a small amount of a sample. Accordingly, a biochip is expected to play a crucial role in the field of bioinformation analysis using genes, proteins and cells and in use of the bioinformation.

According to the completion of human genome project, studies on micro-analysis system such as DNA chip or protein chip for the analysis of genes or proteins are actively undergoing in the overall biological industry. Biochip market is expected to be growing greatly and has been a major target of development in bio-industry in Korea.

Protein chip or peptide chip is a kind of automatic analysis device for simultaneous analysis of tens or thousands of proteins or peptides fixed on a small substrate, which is the next generation biosensor different from DNA chip in analysis mechanism and applying field. Protein chip is a key technique to disclose functions of biomolecules interacting specifically to specific proteins and to develop a novel method for preventing and treating disease, particularly those diseases untreatable so far by the conventional arts, based on functional analysis and network analysis of protein.

Protein chip techniques are largely divided into three categories; protein microarray techniques related to chip production, analysis techniques for quantification of protein interaction to observe proteins fixed on array and application techniques of protein chip. First, protein microarray chip is prepared by different methods according to techniques to analyze protein chip. For example, in the case of using SPR (Surface Plasmon Resonance), proteins have to be fixed on a thin metal film such as a thin gold film. Therefore, a thin gold film construction method and a protein fixation technique have to be developed. In the case of using a fluorescent material, proteins are fixed directly on a slide glass for analysis. So, the proteins have to be labeled with fluorescent materials.

The second core technique, the analysis technique of protein chip includes SPR, mass spectrometry, fluorometry, electrochemical analysis method and nano-imaging technique such as Ellipsometry, which has been developed very competitively so far. Fluorometry based on DNA chip analysis has been most widely used so far, but each method has been recognized to have merits and demerits, and which protein chip analysis method is most appropriate for disease is still undecided.

The last core technique, the application technique of protein chip has the biggest potential for the development and still leaves a lot to discover.

The newly developed techniques related to protein chip are classified into four categories as follows.

(1) A technique to analyze interaction between DNA and protein on the chip by using DNA microarray. On the chip, single-stranded oligonucleotide is converted into double-stranded oligonucleotide, to which specific DNA sequence restriction enzyme is reacted. Then, DNA-protein interaction was examined by measuring digestion. So, this technique is effective in identifying a novel DNA binding protein and in disclosing the characteristics thereof (Bulyk, M. L. et al., Nature Biotechnol., 17:573-577, 1999).

(2) A technique to analyze diverse enzymes including restriction enzyme, peroxidase, phosphatase and protein kinase, and antigen-antibody reaction on the chip (US Patent Publication No. 2002/0055186A1; WO 01/83827A1; Braunwalder A. et al., Anal. Biochem., 234:23-26, 1996; Houseman B. et al., Nature Biotechnol., 20:270-274, 2002; Ruud M. et al., Nature Biotechnol., 18:989-994, 2000). In particular, this technique can be applied in mass-analysis, biochemical analysis, new drug candidate analysis and disease diagnosis by examining protein-protein interaction, kinase-peptide substrate reaction and protein-ligand binding reaction. However, when a kinase specific substrate peptide or a low-molecular weight protein is fixed, bovine serum albumin (BSA) is necessarily added in order to prevent non-specific fixation, which buries the protein fixed thereon. Besides, when different antibodies are fixed on the chip to be reacted with fluorescein labeled antigen mixture, only 60% of the antibodies were quantified and only 23% of the antibodies were qualified (MacBeath G. et al., Science, 289:1760-1763, 2000; Haab B. et al., Genome Biol. 2:research 0004, 2001).

(3) A technique to analyze massive proteins expressed on the chip from cDNA library (WO 01/83827, WO 02/50260). This technique is effective in large scale measurement of biochemical activity of a protein (Heng Zhu, et al., Nature genetics, 26:283-289, 2000).

(4) A method to analyze a sample by using the technique of regulating orientation of biomolecules at molecular level using affinity tag and forming a stable and single layer of biomolecules on the surface of the chip (US Patent Publication No. 2002/0055125A1; U.S. Pat. No. 6,406,921; Paul J. et al., JACS, 122:7849-7850, 2000; RaVi A. et al., Anal. Chem., 73:471-480, 2001; Benjamin T. et al., Tibtech., 20:279-281, 2002). For example, a protein is expressed as a His-tag fusion protein, which is reacted to a chip fixed with Ni-NTA functional group, leading to fixation. The protein is then expressed as an intein fused protein or retains the activity, which not only makes the purification easy but also makes the protein on the chip more stable and active by fixing in a regular direction on the avidin treated chip (Zhu et al., Science, 293:2101-2105, 2001; Marie-Laure L. et al., JACS 124:8768-8769, 2002). And, the protein is expressed on the chip as a fusion protein using a supporter specific protein (calmodulin, etc) and tag (poly cysteine, lysine, histidine, etc) and fixed. The protein is purified by using protein-protein interaction and further used for SPR (surface plasmon resonance) and FACS (fluorescence activated cell sorter) (Hentz et al., Anal. Chem., 68:3939-3944, 1996; Hodneland et al., PNAS, 99:5048-5052, 2002; Kukar et al., Anal. Biochem., 306:50-54, 2002; U.S. Pat. No. 6,117,976).

Kinase is a protein enzyme involved in signal transduction pathways to induce a series of reactions in vivo and hence it is considered to be an effective target for a drug. Kinase provides γ-phosphorylated group to serine, threonine and tyrosine residues of the specific sequence of a target protein from ATP provided in cells, by which it is involved in signal transduction pathway in eukaryotic cells and various diseases (Hunter, T., Cell 100:113-127, 2000; Zhang, Z. Y., Curr. Opin. Chem. Bol. 5:416-423, 2001). The conventional method to study the activity of kinase is the examination of cell membrane using a radio-isotope, but this method is very slow-going and requires a great labor. Another conventional method to measure kinase and its receptor in a large scale is ELISA (enzyme-linked immunosorbent assay) or a method using antibody. ELISA is a comparatively accurate method but takes a lot of time and requires a huge amount of samples. The method using an antibody facilitates mass-analysis but requires high costs and the processes are very complicated.

Promega Co., USA provides a phosphorylation assay kit using a membrane having high absorptiveness to ATP labeled with a radio-isotope and biotinated kinase matrix. However, this kit is limited in mass-analysis. This company also provides an analysis method without using a radio-isotope but using moving difference on electrophoresis caused by changes of net charge of the matrix after phosphorylation of kinase. But, this method requires high costs for mass-analysis. Except for the said four protein chip related techniques, there is no method available for screening the activity of protein kinase in a large scale by using protein chip or peptide chip. Therefore, it is urgently required to develop a new system facilitating accurate and fast analysis with low costs.

The present inventors constructed a biochip for the detection of phosphorylation in Korean Patent Application No. 2007-0070049, in which kinase matrix is integrated on the surface of a substrate directly or as a fusion protein fused to E. coli malic enzyme, but further studies on the biochip using PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) matrix are required.

Thus, the present inventors further studied and constructed a recombinant fusion protein from the fusion of PKC, cdc2-PK or DNA PK matrix with Selenomonas ruminantium membrane protein and then completed this invention by constructing a protein chip on which the recombinant fusion protein is integrated on the surface of a substrate coated with an active group and measuring phosphorylation by the kinase.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a biochip constructed by integrating a recombinant fusion protein prepared by the fusion of a kinase matrix selected from the group consisting of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase) with the elevated protein, Selenomonas ruminantium membrane protein on the surface of a substrate coated with an active group, and a detection kit of phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope, and a detection method of phosphorylation using the same.

The biochip for the detection of phosphorylation and the detection method of phosphorylation using the same of the present invention increase sensitivity by using a radio-isotope, by which a smaller amount of a sample can be used by simpler processes, compared with the conventional method. In addition, the method of the present invention produces a very clear result owing to a simplified procedure excluding blocking process for preventing non-specific fixation on the chip. So, using the biochip of the present invention has advantages of saving time and having economical efficiency. According to the present invention, only a small amount of a sample is required, suggesting that the size of a spot is very small, indicating the number of samples that can be integrated on the surface can be increased, which enables fast analysis of a large amount of samples within a short period. Therefore, the method of the present invention can be effectively used for the analysis of kinase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

FIG. 1 is a diagram showing the nucleotide sequence of Mep45 derived from Selenomonas ruminantium and the amino acid sequence derived therefrom:

Box: promoter, SLH domain, porin homology region; and

Arrow: digestion site by the treatment of proteinase K in the whole cell.

FIG. 2 is a diagram illustrating the cloning process of the recombinant Mep45-kinase matrix fusion protein.

FIG. 3 is a diagram illustrating the phosphorylation of kinase-matrix using [γ-³²P]ATP.

FIG. 4 is a diagram illustrating the phosphorylation of cdc2 protein kinase and peptide matrix using [γ-³²P]ATP:

(A) a: negative control (BSA), b: cdc2 protein kinase (peptide); and

(B) a: negative control (BSA), b: cdc2 protein kinase matrix (Mep45 fusion matrix ).

FIG. 5 is a diagram illustrating the phosphorylation of PKC (protein kinase C) and peptide matrix using [γ-³²P]ATP:

(A) a: negative control (BSA), b: PKC (peptide); and

(B) a: negative control (BSA), b: PKC matrix (Mep45 fusion matrix ).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

To achieve the above object, the present invention provides a biochip constructed by integrating a recombinant fusion protein prepared by the fusion of a kinase matrix having one of the amino acid sequences represented by SEQ. ID. NO: 1-NO: 3 with an elevated protein on the surface of a substrate coated with an active group.

The present invention also provides a detection kit of phosphorylation containing the said biochip and [γ-³²P]ATP.

The present invention further provides a detection method of phosphorylation comprising the following steps:

1) mixing a sample with [γ-³²P]ATP;

2) inducing phosphorylation by treating the biochip with the mixed sample of step 1);

3) washing the biochip of step 2); and

4) Measuring phosphorylation by photo-sensitizing the biochip of step 3).

Hereinafter, the present invention is described in detail.

The present invention provides a biochip constructed by integrating a recombinant fusion protein prepared by the fusion of a kinase matrix having one of the amino acid sequences represented by SEQ. ID. NO: 1-NO: 3 with an elevated protein on the surface of a substrate coated with an active group.

The material of the substrate of the biochip of the present invention is preferably selected from the group consisting of glass, plastic, metal and silicon, and glass is more preferred, but not always limited thereto.

The active group coated on the substrate of the biochip of the present invention plays a role in fixing a peptide and is preferably selected from the group consisting of amine group, aldehyde group, carboxyl group and thiol group, and aldehyde group is more preferred, but not always limited thereto. In fact, almost every active group known to those in the art as an active group capable of fixing a protein molecule on a substrate can be used.

The kinase matrix of the biochip of the present invention is preferably the matrix (AAKIQASFRGHMARKK; SEQ. ID. NO: 1, PKTPKKAKKL; SEQ. ID. NO: 2, or EPPLSQQAFADLWKK; SEQ. ID. NO: 3) for PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase), but not always limited thereto.

The elevated protein of the biochip herein is preferably Selenomonas ruminantium membrane protein (SrMep45), but not always limited thereto.

The kinase matrix herein is fixed on the biochip of the present invention as the elevated protein-matrix fusion protein, but not always limited thereto.

On the biochip of the present invention, the diameter of the integrated spot is preferably 100˜300 μm and the distance between the spots is preferably 300˜500 μm, but not always limited thereto.

The present inventors prepared a recombinant fusion protein using PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) matrix and Selenomonas ruminantium membrane protein. Then a biochip was constructed by fixing the recombinant fusion protein on the surface of a slide glass treated with aldehyde group, the functional group fixable protein, only by spotting method (see FIG. 2).

The biochip of the present invention does not need blocking process which is necessary for the conventional methods. This causes the burial of proteins by the blocker. BSA is used for the prevention of non-specific fixation observed during the fixation of a kinase specific peptide or a low molecular protein. So, using the biochip of the present invention makes the process simple and has advantages of saving time and having economical efficiency.

To confirm the conditions of phosphorylation of kinase-matrix using [γ-³²P]ATP, the biochip prepared above was treated with kinase buffer containing [γ-³²P]ATP and Mep45-kinase matrix fusion protein to induce phosphorylation. Then, phosphorylation was measured by using X-ray film and X-ray fluorescence spectrometer (see FIG. 3). As a result, there was no signal observed on the spot of BSA, the negative control, while signals were observed on the spots of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) matrix and the matrix-SrMep45 fusion protein. The results indicate that PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) matrix and the radio-isotope [γ-³²P]ATP of the present invention can be used for the measurement of phosphorylation (see FIGS. 4 and 5).

The present invention also provides a detection kit of phosphorylation containing the said biochip and [γ-³²P]ATP.

The kit herein can measure phosphorylation of a kinase selected from the group consisting of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase), but not always limited thereto.

Preferably, the kit additionally contains protein kinase as a positive control.

The present invention further provides a detection method of phosphorylation comprising the following steps:

1) mixing a sample with [γ-³²P]ATP;

2) inducing phosphorylation by treating the biochip with the mixed sample of step 1);

3) washing the biochip of step 2); and

4) Measuring phosphorylation by photo-sensitizing the biochip of step 3).

In the above method, the sample of step 1) can be selected from the group consisting of extracts of cells and tissues, fractions or cell culture solutions, cell lysates, crude extracts of cells or tissues, exudates such as urine, sweat, saliva and tear, and body fluids such as blood, plasma, lymph and serum, but not limited thereto and any biological sample known to those in the art can be used.

In this method, the reaction of the sample and the biochip of step 2) is induced in a 30° C. or 37° C. humid chamber for 30 minutes-1 hour, and one hour reaction is more preferred, but not always limited thereto, and the reaction time can be varied according to the matrix-kinase specificity of the sample.

In this method, the measurement of phosphorylation in step 4) is performed by using X-ray film or X-ray fluorescence spectrometer, but not always limited thereto. Sensitization time for the measurement is 12-24 hours, but not always limited thereto, and can be varied according to the matrix-kinase specificity of the sample.

The detection method of phosphorylation of the present invention has advantages of a simple process of chip surface treatment for matrix fixation and fast and easy measurement, compared with the conventional fluorescence ELISA, because the one-pot labeled radio-isotope can be detected as it is. According to the conventional fluorescence detection method, a specific amino acid of matrix has to be phosphorylated first, and then the phosphorylated amino acid region is reacted with a secondary antibody labeled with a fluorescent material, which is an indirect detection method that cannot provide precise quantification at a satisfactory level. However, the detection method of phosphorylation of the present invention is characterized by direct labeling of a radio-isotope to the matrix, so that the method not only facilitates precise quantification of detection result but also facilitates detection with a minimum amount with high sensitivity.

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

EXAMPLE 1 Preparation of Elevated Protein-Kinase Fusion Protein

<1-1> Cloning of SrMep45-Matrix Fusion Protein

To prepare SrMep45 (Selenomonas ruminantium membrane protein)-matrix fusion protein, the PCR product obtained by PCR amplification with chromosome DNA of Selenomonas ruminantium subsp. lactilytica, ATCC 19205) (Kanegasaki, S., and Takahashi, H., J. Bacterial. 93, 456-463, 1967) and plasmid were cloned (FIG. 1).

For the cloning of each matrix (AAKIQASFRGHMARKK; SEQ. ID. NO: 1, PKTPKKAKKL; SEQ. ID. NO: 2, or EPPLSQQAFADLWKK; SEQ. ID. NO: 3) for PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase), PCR was performed using pSrMep45 as a template with primers PKC-Fw-Nde (5-CATCATATGGCTGCTAAAATTCAAGCTTCTTTTCGTGGTCATATGGCTCGTAAAAAAGCTAGC AACCCGTTCTCCGATG-3′; SEQ. ID. NO: 4), PKC-Rv-Bam (5′-GACGGATCCTTATTTTTTACGAGCCATATGACCACGAAAAGAAGCTTGAATTTTAGCAGCGAA GAAGAACTGAACGCGACCGAAG-3′; SEQ. ID. NO: 5), cdc2-MP-Fw-Nde (5′-CATCATATGCCTAAAACTCCTAAAAAAGCTAAA AAACTTGCTAGCAACCCGTTCTCCGATG-3′; SEQ. ID. NO: 6), cdc2-MP-Rv-Bam (5′-GACGGATCCTTAAAGTTTTTTAGCTTTTTTAGGAGTTTTAGGGAAGAAGAACTGAACGCGACC GAAG-3′; SEQ. ID. NO: 7), DNA-PK-MP-Fw-Nde (5′-CATCATATGGAACCTCCTCTTTCTCAACAAGCTTTTGCTGATCTTTGG AAAAAAGCTAGCAACCCGTTCTCCGATG-3′; SEQ. ID. NO: 8) and DNA-PK-MP-Rv-Bam (5′-GACGGATCCTTATTTTTTCCAAAGATCAGCAAAAGCTTGTTGAGAAAGAGGAGGTTCGAAGAA GAACTGAACGCGACCGAAG-3′; SEQ. ID. NO: 9) (Table 1). The PCR was performed using 2.0 unit Taq DNA polymerase (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl₂, 0.01% gelatin, 0.1% Triton X-100), 0.4 mM dNPT (deoxyribonucleotide triphosphate) and the reaction mixture containing the said primer set with Palm-cycler (Corbett Life Science, USA) as follows: at 94° C. for 5 minutes (1 cycle); and at 94° C. for 1 minute, at 55° C. for 1 minute, and at 72° C. for 1 minutes (30 cycles); and then at 72° C. for 5 minutes (1 cycle). The PCR product was electrophoresed on 0.8% agarose gel, followed by staining with SYBR Green I. The amplified right size DNA was purified by using Gel Extraction Spin Column (Bio-Rad Lab., USA). The amplified DNA was ligated to plasmid pGEM-T Easy vector, followed by sequencing. The subcloned sample was digested with NdeI and BamI, which was ligated to pET-28a predigested with the same enzymes. E. coli BL21(DE3) was transformed with the vector. As a result, a new strain for the production of the recombinant Mep45-kinase matrix fusion protein was generated (FIG. 2).

TABLE 1 Target kinase matrix and primer for cloning thereof Matrix Primer Sequence SEQ. ID. NO: PKC (Protein PKC-Fw-Nde 5′-CAT CAT ATG GCT GCT AAA SEQ. ID. NO: 4 Kinase C) ATT CAA GCT TCT TTT CGT GGT matrix CAT ATG GCT CGT AAA AAA GCT AGC AAC CCG TTC TCC GAT G-3′ PKC-Rv-Bam 5′-GAC GGA TCC TTA TTT TTT SEQ. ID. NO: 5 ACG AGC CAT ATG ACC ACG AAA AGA AGC TTG AAT TTT AGC AGC GAA GAA GAA CTG AAC GCG ACC GAA G-3′ cdc2-PK cdc2-MP-Fw-Nde 5′-CAT CAT ATG CCT AAA ACT SEQ. ID. NO: 6 (cdc2 CCT AAA AAA GCT AAA AAA CTT Protein GCT AGC AAC CCG TTC TCC GAT Kinase) G-3′ matrix cdc2-MP-Rv-Bam 5′-GAC GGA TCC TTA AAG TTT SEQ. ID. NO: 7 TTT AGC TTT TTT AGG AGT TTT AGG GAA GAA GAA CTG AAC GCG ACC GAA G-3′ DNA-PK (DNA- DNA-PK-MP-Fw-Nde 5′-CAT CAT ATG GAA CCT CCT SEQ. ID. NO: 8 dependent CTT TCT CAA CAA GCT TTT GCT Protein GAT CTT TGG AAA AAA GCT AGC Kinase) AAC CCG TTC TCC GAT G-3′ matrix DNA-PK-MP-Rv-Bam 5′-GAC GGA TCC TTA TTT TTT SEQ. ID. NO: 9 CCA AGG ATC AGC AAA AGC TTG TTG AGA AAG AGG AGG TTC GAA GAA GAA CTG AAC GCG ACC GAA G-3′

Vectors encoding the final Mep45-matrix fusion protein were pMep45PKC1, pMep45PKC2, pMep45cdc21, pMep45cdc22, pMep45DPK1 and pMep45DPK2, as shown in Table 2.

TABLE 2 Cloned vector for each Mep45-matrix fusion protein Matrix Cloned vector Structure PKC Matrix pSrMep45PKC1 •-□ pSrMep45PKC2 •-□-• cdc2-PK Matrix pSrMep45cdc21 ♦-□ pSrMep45cdc22 ♦-□-♦ DNA-PK Matrix pSrMep45DPK1 ▴-□ pSrMep45DPK2 ▴-□-▴ □: SrMep45; •: PKC matrix; ♦: cdc2-PK matrix; and ▴: DNA-PK matrix.

<1-2> Production and Purification of Recombinant Mep45-Kinase Matrix Fusion Protein

The strain prepared in Example <1-1> was cultured in 200 ml of LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L) in a 500 ml Erlenmeyer flask at 30° C. with 200 rpm. The antibiotic kanamycin was added at the final concentration of 50 μg/ml. The cells were cultured until O.D.₆₆₀ reached 0.4. IPTG was added at the final concentration of 0.1 mM, followed by further culture at 30° C. with 200 rpm for 3 hours. Upon completion of the culture, the cells were collected (8,000 rpm, 10 minutes, 4° C.). The collected cells were suspended in PBS (200 mM NaCl, 3 mM KCl, 2 mM KH₂PO₄, 1 mM Na₂HPO₄, pH 7.5) and lysed using ultra-sonicator. Cell debris was eliminated by centrifugation. The recombinant Mep45-kinase matrix fusion protein was purified by using Ni-chelating resin (GE Healthcare, Sweden) to 6-histidine tag. The purified protein was quantified according to Bradford method using BSA (bovine serum albumin) as a standard.

The mass of the SrMep45-matrix protein was approximately 45 kDa, which was bound to the aldehyde treated slide to protect active site of the matrix being apt to be buried by BSA used in blocking stage.

EXAMPLE 2 Construction of Biochip

To fix matrix on the aldehyde treated slide glass (Nuricell Inc., Korea), 0.1 mg/ml of the Mep45-kinas matrix fusion protein or 1.25 μg/ml of peptide matrix (Promega, Madison, Wis.) was integrated. Particularly, the recombinant Mep45-kinase matrix fusion protein solution (10% glycerol, PBS, pH 7.5) was prepared at the concentration of 0.1 mg/ml, and this matrix solution was integrated on the aldehyde treated slide glass at the spot intervals of 300 μm by using microarray device (Genetix Ltd, UK). The size of the spot was regulated to be 300 μm. The integrated biochip was reacted in a humid chamber at room temperature for one hour, leading to fixation.

EXAMPLE 3 Confirmation of Phosphorylation Conditions for Kinase-Matrix using [γ-³²P]ATP

The biochip constructed in Example 2 was washed three times with PBS (200 mM NaCl, 3 mM KCl, 2 mM KH₂PO₄, 1 mM Na₂HPO₄, pH 7.5), followed by reaction of the matrix and kinase on the chip. Particularly, the chip was washed with kinase buffer (40 mM Tris-HCl, 20 mM MgCl₂, 0.1 mg/ml BSA, pH 7.5) once. Then, 50 μl of kinase reaction solution (kinase buffer containing 100 μM ATP, [γ-³²P]ATP (0.1˜0.6 μCi) (GE Healthcare Life Sciences, UK) and 0.01˜50 unit/ml recombinant Mep45-kinase matrix fusion protein) was distributed on the surface of the biochip. The biochip was covered with cover well, followed by reaction for one hour. One hour later, the biochip was washed with washing buffer three times, followed by washing again with PBS. Centrifugation was performed at 200×g for one minute to eliminate remaining moisture completely. The reacted biochip was sensitized on X-ray film or screen of bioimage analyzer BAC1500 (Fuji Photo Film, Tokyo) for 6-14 hours, followed by measurement of phosphorylation by kinase (FIG. 3).

<3-1> Confirmation of Phosphorylation of cdc2 Protein Kinase-Matrix

1.0 mg/ml of PKTPKKAKKL and Mep45 fusion matrix were integrated on the aldehyde treated slide glass, followed by phosphorylation using 10 unit/ml of cdc2 protein kinase and 0.1 μCi/μl of [γ-³²P]ATP. Phosphorylation was measured by using X-ray film or X-ray fluorescence spectrometer. BSA (Bovine Serum Albumin) was used for the negative control.

As a result, no signal was detected on the spot of BSA (negative control), while clear signal was detected on the spot of PKTPKKAKKL matrix (FIG. 4A) and Mep45 fusion matrix (FIG. 4B), suggesting that the radio-isotope [γ-³²P]ATP could be effectively applied for the primary detection of phosphorylation (FIG. 4).

<3-2> Confirmation of Phosphorylation of PKC-Matrix

0.3 mg/ml of AAKIQASFRGHMARKK and Mep45 fusion matrix were integrated on the aldehyde treated slide glass, followed by phosphorylation using 10 unit/ml of PKC and 0.1 μCi/μl of [γ-³²P]ATP. Phosphorylation was measured by using X-ray film or X-ray fluorescence spectrometer. BSA (Bovine Serum Albumin) was used for the negative control.

As a result, no signal was detected on the spot of BSA (negative control), while clear signal was detected on the spot of AAKIQASFRGHMARKK matrix (FIG. 5A) and Mep45 fusion matrix (FIG. 5B), suggesting that the radio-isotope [γ-³²P]ATP could be effectively applied for the primary detection of phosphorylation (FIG. 5).

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims. 

1. A biochip constructed by integrating a recombinant fusion protein prepared by the fusion of a kinase matrix having one of the amino acid sequences represented by SEQ. ID. NO: 1-NO: 3 with an elevated protein on the surface of a substrate coated with an active group.
 2. The biochip according to claim 1, wherein the substrate is selected from the group consisting of glass, plastic, metal and silicon.
 3. The biochip according to claim 1, wherein the active group is selected from the group consisting of amine group, aldehyde group, carboxyl group and thiol group.
 4. The biochip according to claim 1, wherein the kinase is selected from the group consisting of PKC (protein kinase C), cdks (cyclin-dependent protein kinase) and DNA-PK (DNA-dependent protein kinase).
 5. The biochip according to claim 1, wherein the elevated protein is Selenomonas ruminantium membrane protein.
 6. The biochip according to claim 1, wherein the diameter of the integrated spot is 100˜300 μm and the distance between the spots is 300˜500 μm.
 7. A kit for the detection of phosphorylation containing the biochip of claim 1 and [γ-³²P]ATP.
 8. The kit for the detection of phosphorylation according to claim 7, wherein the kit additionally contains protein kinase as a positive control.
 9. A detection method of phosphorylation comprising the following steps: 1) mixing a sample with [γ-³²P]ATP; 2) inducing phosphorylation by treating the biochip with the mixed sample of step 1); 3) washing the biochip of step 2); and 4) Measuring phosphorylation by photo-sensitizing the biochip of step 3).
 10. The detection method of phosphorylation according to claim 9, wherein the sample of step 1) is selected from the group consisting of cell culture solutions, cell lysates, crude extracts of cells or tissues, exudates such as urine, sweat, saliva and tear, and body fluids such as blood, plasma, lymph and serum.
 11. The detection method of phosphorylation according to claim 9, wherein the biochip of step 2) is not treated with blocking solution. 12 The detection method of phosphorylation according to claim 9, wherein the induction of step 2) is performed at 30° C. or at 37° C. for 30˜60 minutes.
 13. The detection method of phosphorylation according to claim 9, wherein the photo-sensitization of step 4) is performed by using X-ray film or X-ray fluorescence spectrometer. 